Thioredoxin system is a cellular redox system present in all previously characterized organisms. Thioredoxin reductase (TR) controls the redox state of thioredoxin, which in turn regulates numerous cellular processes. The function and catalytic activity of mammalian TR is dependent on a selenocysteine residue, whose biosynthesis in turn is affected by the availability of dietary selenium. Two mammalian TR isozymes (TR2 and TR3), in addition to the previously characterized TR1, have recently been identified in humans and mice. The three TR isozymes exhibit extensive homology but differ in expression patterns and cellular locations. Additional forms of TR isozymes have been identified that are generated by alternative first exon splicing resulting in remarkable heterogeneity of mammalian TR preparations. The purpose of the proposed study is to functionally characterize mammalian TR isozymes, with the emphasis on the role of the selenocysteine residue and on the roles of TR isozymes in redox regulation of cellular processes. A combination of biochemical and cell biology approaches will be used to address the following specific questions (specific aims): 1) what are the reaction mechanisms of TR isozymes? We will characterize wild-type and mutant TR isozymes and test the hypothesis that the selenocysteine center transfers reducing equivalents from the N-terminal redox center of TR to a thiol oxidoreductase substrate; 2) what are the tissue expression patterns, cellular locations and regulation of expression of TR isozymes and their alternatively spliced forms; and 3) what are the roles of TR isozymes in cellular redox regulation and their place in the hierarchy of mammalian selenoproteins?